Eukaryotic tRNAs are transcribed as precursors and must be processed at both ends before aminoacylation and translation. A 5' end leader is cleaved off by RNase P and a 3' end trailer can be endonucleolytically removed by 3'-tRNase. Finally, tRNA nucleotidyltransferase (NTase) adds CCA to the 3' end left by 3'-tRNase. Homologs of these tRNA end-processing enzymes have been identified and partially characterized in Drosophila melanogaster. We propose to investigate pre-tRNA end-processing by all three enzymes, taking advantage of the biology and fully sequenced Drosophila genome, as follows: Aim 1. RNAi, a powerful, innovative tool for knocking down gene expression in vivo, will be used against RNase P and 3'-tRNase to investigate reaction order and identity of nuclear and mitochondrial forms of these enzymes. Aim 2. The 3'-tRNase anti-determinant hypothesis (that tRNA with CCA at its mature 3' end does not recycle through 3'-tRNase) will be further investigated. Aim 3. Stoichiometric complexes of 3'-tRNase and NTase with substrates, products and substrate analogs will be analyzed to better understand the sites and domains required for substrate recognition, binding and catalysis. Aim 4. The solution of 3'-tRNase structure, both free and complexed with tRNA, will be undertaken by X-ray diffraction crystallography. This coordinated, comprehensive study of D. melanogaster pre-tRNA end-processing is made possible by an established network of collaborations with world-class scientists.